The overall focus of the Project, Molecular Interactions of Lymphoid Cell Receptors, is a broad one, based on the crucial importance of the recognition of MHC molecules by many receptors of the innate and adaptive immune systems as well as interactions of other receptors such as TLRs (Toll like receptors) and LAIR-1 (the leukocyte-associated inhibitory receptor). Our major interest is focused on MHC molecules and their interactions with T cell receptors, T cell coreceptors, CD8 and CD4, and natural killer (NK) cell receptors. Because of our particular expertise in measurement of interactions of recombinant lymphoid receptors and antibodies using biophysical methods, such as surface plasmon resonance, we often collaborate with colleagues who have other molecules of interest. Thus our work with TLRs and LAIR-1 derives from such collaborative relationships. Our general approach is to exploit cloned genes to express recombinant forms of the molecules of interest, to analyze their function using in vitro cellular assays, to analyze their biophysical properties using state-of-the-art methodologies such as surface plasmon resonance and analytical ultracentrifugation, and to understand the three-dimensional structural basis of their function with high-resolution X-ray crystallographic structural models. Our collaborative studies examined the cooperative interaction of TLR3 with itsdouble stranded RNA ligand. In addition, using both direct binding methods and computer graphics molecular modeling approaches we explored the binding of LAIR-1 with collagen and explained the defect in LAIR-1 function resulting from a point mutation. A major accomplishment of in the past year and a half has been the determination of the X-ray structure of the multimolecular five-subunit complex consisting of MHC/beta-2 microglobulin/peptide/CD8alpha/CD8beta. Although the structure of the first MHC-I molecule was determined in 1987, and the structure of the human CD8 alpha/alpha homodimer was determined in 1992, and structures of CD8 alpha/alpha in complex with MHC-I and of mouse CD8 alpha/beta unliganded have been reported, there has been no successful determination of the structure of the CD8 alpha/beta heterodimer in complex with MHC-I. This is of considerable importance because CD8 alpha/beta, not CD8 alpha/alpha, is the major functional molecule expressed on mature CD8+ cells. Rational strategies to develop highly efficient CTL for killing tumors would depend on knowledge of the MHC/beta2-m/CD8alpha/beta complex. Binding and quantitative functional studies from several laboratories indicate that CD8 achieves its coreceptor function by improving the functional readout of T cell activation, but not by increasing avidity. That is, the measured affinities of CD8 alpha/alpha and CD8 alpha/beta for MHC-I/peptide complexes have been measured to be about the same. Extensive binding and functional analyses of a battery of CD8 alpha/beta mutants in several laboratories have led to the rather unsatisfying conclusion that CD8 alpha/beta may not bind to MHC-I in a single conformation, but rather may assume one of several different distinct conformations, accounting for several ambiguous results derived from the mutagenesis studies. To address the issue of the nature of the conformation of CD8 alpha/beta when bound to an MHC-I molecule, we explored several different approaches to engineering CD8 alpha/beta for structural studies, and finally succeeded with mouse CD8 alpha and CD8 beta extracellular domains engineered for expression as inclusion bodies in E. coli. These were then solubilized and refolded with minor variations of our standard methods, and stable, disulfide-linked CD8 alpha/beta heterodimers were produced, purified free from contaminating homodimers, and used in co-crystallization trials with bacterially expressed, refolded, highly purified H-2Dd/mouse beta2m/P18-I10 complexes. Crystals were obtained, the best of which diffracted to 2.6 Angstroms. The results of this structure indicate that the CD8beta chain is in the membrane distal position, and reveal numerous details of the interaction of the CD8 heterodimer with the MHC-I molecule.